Mitosis is initiated by the activation of cyclin B1-Cdk1 and the translocation of the active complexes to the nucleus. The all-or-none, irreversible nature of mitosis arises from a bistable trigger circuit composed of interlinked positive and double-negative feedback loops where active cyclin B1-Cdk1 promotes cyclin B1-Cdk1 activation, and nuclear cyclin B1-Cdk1 promotes cyclin B1-Cdk1 translocation. This systems-level behavior depends upon the kinetic properties of the individual components of the feedback loops, including the shapes of their response functions. During the last funding period, we showed that the multisite phosphorylation of Wee1A and Cdc25C allows them to generate highly ultrasensitive response, which in turn makes the bistability of the mitotic trigger possible. Here we propose to build upon these discoveries to determine how multisite phosphorylation leads to the ultrasensitive regulation of the key Cdk1 substrate and regulator Wee1A, and to examine the temporal order of protein dephosphorylation during mitotic exit. There are three Specific Aims: Aim 1. The role of Cks proteins in the regulation of Cdk phosphorylation. Mitotic Cdk1 complexes consist of three proteins: the Cdk1 protein kinase, the allosteric activator cyclin B, and a third small protein referred to as Suc1 in S. pombe, Cks1 in S. cerevisiae, and Cks1 or 2 in vertebrates, whose function is more enigmatic. We have recently found that Xenopus Cks2 acts as a bivalent adaptor, binds independently to both Cdk1 and phospho-Wee1A, and builds a substantial threshold into the switch-like response of Wee1A to Cdk1. Here we aim to explore the generality of this phenomenon and examine the regulation of Cks2 by phosphorylation. Aim 2. The mechanism of Wee1A inactivation. We have shown that the phosphorylation of Wee1A at T53 primes the protein for subsequent phosphorylations at T104 and T150, and that these phosphorylations result in Wee1A inactivation. Here we propose a series of biophysical and structural studies aimed at understanding how T104/T150 phosphorylation affects the Wee1A kinase domain. Aim 3. Mechanisms of temporal organization in mitotic exit. Mitotic exit is a highly organized cell biological process. Here we propose to describe how the dephosphorylation of mitotic phosphoproteins is organized temporally, and determine what mechanisms allow the dephosphorylation to proceed in an orderly fashion.